NOCICEPTRA: Gene and microRNA Signatures and Their Trajectories Characterizing Human iPSC-Derived Nociceptor Maturation.

Nociceptors are primary afferent neurons serving the reception of acute pain but also the transit into maladaptive pain disorders. Since native human nociceptors are hardly available for mechanistic functional research, and rodent models do not necessarily mirror human pathologies in all aspects, human induced pluripotent stem cell-derived nociceptors (iDN) offer superior advantages as a human model system. Unbiased mRNA::microRNA co-sequencing, immunofluorescence staining, and qPCR validations, reveal expression trajectories as well as miRNA target spaces throughout the transition of pluripotent cells into iDNs. mRNA and miRNA candidates emerge as regulatory hubs for neurite outgrowth, synapse development, and ion channel expression. The exploratory data analysis tool NOCICEPTRA is provided as a containerized platform to retrieve experimentally determined expression trajectories, and to query custom gene sets for pathway and disease enrichments. Querying NOCICEPTRA for marker genes of cortical neurogenesis reveals distinct similarities and differences for cortical and peripheral neurons. The platform provides a public domain neuroresource to exploit the entire data sets and explore miRNA and mRNA as hubs regulating human nociceptor differentiation and function.

Presynaptic α2δ subunits are key organizers of glutamatergic synapses.

In nerve cells the genes encoding for α2δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α2δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α2δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α2δ isoforms as synaptic organizers is highly redundant, as each individual α2δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α2δ-2 and α2δ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α2δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α2δ implicates α2δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α2δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density.

Phenotypic Characterization and Brain Structure Analysis of Calcium Channel Subunit α2δ-2 Mutant (Ducky) and α2δ Double Knockout Mice.

Auxiliary α2δ subunits of voltage-gated calcium channels modulate channel trafficking, current properties, and synapse formation. Three of the four isoforms (α2δ-1, α2δ-2, and α2δ-3) are abundantly expressed in the brain; however, of the available knockout models, only α2δ-2 knockout or mutant mice display an obvious abnormal neurological phenotype. Thus, we hypothesize that the neuronal α2δ isoforms may have partially specific as well as redundant functions. To address this, we generated three distinct α2δ double knockout mouse models by crossbreeding single knockout (α2δ-1 and -3) or mutant (α2δ-2/ducky) mice. Here, we provide a first phenotypic description and brain structure analysis. We found that genotypic distribution of neonatal litters in distinct α2δ-1/-2, α2δ-1/-3, and α2δ-2/-3 breeding combinations did not conform to Mendel's law, suggesting premature lethality of single and double knockout mice. Notably, high occurrences of infant mortality correlated with the absence of specific α2δ isoforms (α2Δ-2 > α2δ-1 > α2δ-3), and was particularly observed in cages with behaviorally abnormal parenting animals of α2δ-2/-3 cross-breedings. Juvenile α2δ-1/-2 and α2δ-2/-3 double knockout mice displayed a waddling gate similar to ducky mice. However, in contrast to ducky and α2δ-1/-3 double knockout animals, α2δ-1/-2 and α2δ-2/-3 double knockout mice showed a more severe disease progression and highly impaired development. The observed phenotypes within the individual mouse lines may be linked to differences in the volume of specific brain regions. Reduced cortical volume in ducky mice, for example, was associated with a progressively decreased space between neurons, suggesting a reduction of total synaptic connections. Taken together, our findings show that α2δ subunits differentially regulate premature survival, postnatal growth, brain development, and behavior, suggesting specific neuronal functions in health and disease.

Presynaptic α2δ-2 Calcium Channel Subunits Regulate Postsynaptic GABAA Receptor Abundance and Axonal Wiring.

Presynaptic α2δ subunits of voltage-gated calcium channels regulate channel abundance and are involved in glutamatergic synapse formation. However, little is known about the specific functions of the individual α2δ isoforms and their role in GABAergic synapses. Using primary neuronal cultures of embryonic mice of both sexes, we here report that presynaptic overexpression of α2δ-2 in GABAergic synapses strongly increases clustering of postsynaptic GABAARs. Strikingly, presynaptic α2δ-2 exerts the same effect in glutamatergic synapses, leading to a mismatched localization of GABAARs. This mismatching is caused by an aberrant wiring of glutamatergic presynaptic boutons with GABAergic postsynaptic positions. The trans-synaptic effect of α2δ-2 is independent of the prototypical cell-adhesion molecules α-neurexins (α-Nrxns); however, α-Nrxns together with α2δ-2 can modulate postsynaptic GABAAR abundance. Finally, exclusion of the alternatively spliced exon 23 of α2δ-2 is essential for the trans-synaptic mechanism. The novel function of α2δ-2 identified here may explain how abnormal α2δ subunit expression can cause excitatory-inhibitory imbalance often associated with neuropsychiatric disorders.SIGNIFICANCE STATEMENT Voltage-gated calcium channels regulate important neuronal functions such as synaptic transmission. α2δ subunits modulate calcium channels and are emerging as regulators of brain connectivity. However, little is known about how individual α2δ subunits contribute to synapse specificity. Here, we show that presynaptic expression of a single α2δ variant can modulate synaptic connectivity and the localization of inhibitory postsynaptic receptors. Our findings provide basic insights into the development of specific synaptic connections between nerve cells and contribute to our understanding of normal nerve cell functions. Furthermore, the identified mechanism may explain how an altered expression of calcium channel subunits can result in aberrant neuronal wiring often associated with neuropsychiatric disorders such as autism or schizophrenia.

Emerging evidence for specific neuronal functions of auxiliary calcium channel α2δ subunits.

In nerve cells the ubiquitous second messenger calcium regulates a variety of vitally important functions including neurotransmitter release, gene regulation, and neuronal plasticity. The entry of calcium into cells is tightly regulated by voltage-gated calcium channels, which consist of a heteromultimeric complex of a pore forming α1, and the auxiliary ß and α2δ subunits. Four genes (Cacna2d1-4) encode for the extracellular membrane-attached α2δ subunits (α2δ-1 to α2δ-4), out of which three isoforms (α2δ-1 to -3) are strongly expressed in the central nervous system. Over the years a wealth of studies has demonstrated the classical role of α2δ subunits in channel trafficking and calcium current modulation. Recent studies in specialized neuronal cell systems propose roles of α2δ subunits beyond the classical view and implicate α2δ subunits as important regulators of synapse formation. These findings are supported by the identification of novel human disease mutations associated with α2δ subunits and by the fact that α2δ subunits are the target of the anti-epileptic and anti-allodynic drugs gabapentin and pregabalin. Here we review the recently emerging evidence for specific as well as redundant neuronal roles of α2δ subunits and discuss the mechanisms for establishing and maintaining specificity.